In recent years, a cell culture technology has been developed in which cells, tissues, microorganisms or the like are cultured under an artificial environment, and has been actively used in the medical fields including the production of biological medicines, the gene therapy or regenerative medical therapy and the immuno-cell therapy.
This cell culture technology has already been put into practice in a number of applications where a large amount of cells are required to be cultured efficiently, such as the production of monoclonal antibodies or the regeneration of a skin, and has become a technology of crucial importance.
Under such circumstances, with an aim of culturing efficiently a large amount of cells, various cell culture apparatuses have heretofore been proposed.
For example, the cell culture equipment disclosed in Patent Document 1 has a configuration in which a container main body is provided with a cell culture bag and a prohibiting member for prohibiting the circulation of a culture medium in the culture bag. In this technology, the culture region is divided by means of this prohibiting member, thus enabling gradual extension of the culture region with the proliferation of cells.
By using such cell culture equipment, culture can be conducted continuously within the same culture bag from start to end.
The equipment for in-vitro proliferation disclosed in Patent Document 2 is provided with a series of culture sub-compartments each having a culture space and a regulating means which variably permits or precludes medium intercommunication between sub-compartments, in which a culture region is gradually extended with the proliferation of cells.
With this technology, a sufficiently small-scale starting environment for ensuring viability of the cells, and an enlarging environment can be provided, whereby a high cell number cell masses can be cultured without the risk of transfer contamination economically while saving time.
The culture apparatus disclosed in Patent Document 3 is a culture apparatus for culturing cells which grow along the bottom surface thereof, in which the bottom surface area can be enlarged with the growth of cells.
Therefore, by this technology, cells can be proliferated efficiently in a single container.
However, in these conventional cell culture apparatuses, no special contrivance is made on a culture medium which is to be added when the culture region is extended.
For example, Patent Document 1 states that, if cells proliferate to a desired number in a culture region partitioned by a first prohibiting member, the bag is then partitioned by means of a second partitioning member, and the amount of a culture medium is determined according to the proliferation capability of cells being cultured such that the cell density can be maintained at an appropriate level when the culture region is extended. Patent Document 1 states that the amount of a culture medium is generally determined such that the cell density at the time of starting culture or at the time of extending a culture region becomes about 1×105/ml, and that the cell density may be appropriately determined in accordance with the type of cells used, the characteristics of proliferation or the purpose of culture.
In Patent Document 1, no clear statement is made on how to adjust the components of a culture medium to be added. Therefore, it appears that an optimum culture medium which is used in the original culture medium is added.
However, the culture environment is gradually deteriorated with the proliferation of cells. Therefore, when a culture medium which is the same as the original culture medium is added, although the culture environment is improved immediately after the addition, it is impossible to maintain the environment of the original culture medium.
That is, as a result of cell proliferation, the amount of dissolved oxygen in a culture medium is decreased and the amount of dissolved carbon dioxide in a culture medium is increased. In addition, the pH of the culture medium is gradually decreased by the generation of lactic acid or for other reasons. If a culture medium having the same conditions as those of the original culture medium is added to such a culture medium in the same quantity, for example, it is impossible to allow the entire culture medium to recover the initial optimum environment.
Such a problem also occurs when implementing the inventions disclosed in Patent Documents 2 and 3.
Patent Document 1 states that a bag is put in a carbon dioxide gas culture apparatus, the bag is allowed to stand in a gas atmosphere which is required for the culture, and the conditions such as culture temperature, culture time, pH and carbon dioxide concentration are set in accordance with the type of cells to be used.
Patent Document 2 states that, when the cell growth and viability reaches its limit in the first sub-compartment, the contents thereof (cells, medium) are transferred to the next serial sub-compartment, and, in the next sub-compartment, the cell mass from the first sub-compartment is provided with the conditions required for a further increase with viability being retained according to the culture medium added therein, the volume, or the like.
Furthermore, Patent Document 3 states that, by maintaining menchymal stem cells and a culture medium, which have been mixed, at culture conditions such as a predetermined temperature and a carbon dioxide concentration, cells are subject to secondary culture for a prescribed period of time under fixed culture conditions.
In these conventional technologies, as mentioned above, no consideration is made on component conditions of a culture medium to be added, and a culture medium having the same conditions as those of the original medium is added, and culture conditions such as temperature and CO2 concentration are adjusted after the addition.
Patent Document 1: JP-A-2000-125848
Patent Document 2: Japanese Patent No. 2981684
Patent Document 3: JP-A-2004-89136